Nitric oxide serves as a messenger in signal transduction systems. Reaction of nitric oxide with endogenously generated superoxide anion leads to formation of the highly toxic peroxynitrite. Reaction of peroxynitrite with tyrosine residues of proteins leads to stable nitrotyrosine derivatives. Because nitration of tyrosine precludes its phosphorylation by tyrosine kinases, the nitration of tyrosine residues involved in signal transduction pathways could seriously compromise the regulation of metabolic processes. To determine the effect of nitration on a regulatory enzyme, studies were made using Escherichia coli glutamine synthetase (GS), an enzyme whose activity is under strict control by adenylylation of a unique tyrosine residue in each subunit of the enzyme. As noted in last year's report, treatment of the unadenylylated enzyme with peroxynitrite leads to changes similar to those provoked by adenylylation of the enzyme; whereas treatment of the adenylylated enzyme with peroxynitrite leads to complete loss of activity. In continuing studies, it was established that the ionic strength, pH, and iron-chelates affect the ability of peroxynitrite to nitrate GS. At acid pH (pH 5.5-6.5.), nitration of 1.0 tyrosine residue per subunit (12 per GS dodecamer) converts the enzyme to a form that mimics the form produced by adenylylation, whereas at pH 7.0-8.0 only 0.5 residues per subunit (6 per dodecamer) are required. In addition, treatment with peroxynitrite leads to conversion of 6 of the 16 methionine residues per subunit to methionine sulfoxide residues. Oxidation of GS with hydrogen peroxide, which does not modify tyrosine residues, also led to the oxidation of 8 methionine residues per GS dodecamer. This caused a small but significant effect on the apparent state of adenylylation of the enzyme. Thus, the cytotoxicity of peroxynitrite might be due, in part, to the modification of tyrosine and methionine residues of proteins involved in regulation of metabolic signal transduction networks.